Major causes of retinal photoreceptor degeneration include retinitis pigmentosa (RP), age-related macular degeneration (ARMD), diabetic retinopathy and other diseases. Approximately one in three thousand, or three million people worldwide, suffer from retinitis pigmentosa (RP), a genetic condition that leads to photoreceptor degeneration and eventually blindness. The rate and severity of photoreceptor degeneration is variable and highly dependant on the mutation itself. Over fifty genes may be affected (Hartong et al. Lancet 368:1795-1809; 2006). To date, little treatment is available for RP patients. Ongoing trials that focus on neuroprotective agents (e.g. ciliary neurotrophic factor) or gene addition therapy (introducing the “non-mutated” gene), which aim to correct acquired or hereditary gene deficiencies to the natural functional gene, have so far shown only marginal success. Given that the adult retina has no ability to generate new photoreceptors after photoreceptor loss, gene addition therapy is only useful as long as photoreceptor loss is small and mainly slows down or stabilizes the early condition.
An alternative approach employed in recent experimental studies is to render the remaining photoreceptors or surviving inner retinal neurons light-sensitive through transgenic expression of a light-sensitive protein.
In US 2009/0088399 and US 2010/0015095 it is proposed to introduce the light-gated algal ion-channel channelrhodopsin-2 (ChR2) into the inner retina of patients suffering from photoreceptor cell degeneration This renders the naturally light-insensitive inner retinal cells, such as bipolar or amacrine cells, light-sensitive and capable of detecting visual information, which is subsequently relayed to the brain without receiving input from photoreceptors.
Similarly, in US 2005/0208022 and US 2009/0208462 it is proposed to introduce a photoreceptive protein such as an opsin (including melanopsin) or cytochromes into the inner retinal neurons including amacrine, horizontal and bipolar cells of patients suffering from photoreceptor degeneration.
The approach to express ChR2 in inner retinal neurons holds considerable promise and is currently tested in non-human primates (Fradot M et al. Human Gene Therapy 22(5), 587-593; 2011) and isolated human retinas (Ivanova E et al. Opthalmol Vis Sci 51(10), 5288-5296, 2010), raising hope for clinical trials in the near future.
In recent years retinal gene-replacement therapy using recombinant Adeno-associated virus (rAAV) has been successful and has reached final clinical trials. In particular, Bainbridge and colleagues used rAAV to replace the defective retinal pigment epithelium-specific 65-kDa protein gene (RPE65). A deficiency in the RPE65 protein renders photoreceptors unable to respond to light, as it is required for the recycling of the chromophore, i.e. the conversion of all-trans retinal to 11-cis retinal (Bainbridge J W B et al., N Engl J Med 358(21), 2231-2239; 2008). Gene therapy is therefore a promising therapeutic approach to correct for visual deficiencies by the introduction of suitable genes into retinal neurons.
The currently available light-activatable proteins that could be used in gene therapy to compensate for the loss of photoreceptor cells, however, still hold a number of substantial drawbacks: 1) Artificial expression of foreign, invertebrate or algal proteins, e.g. ChR2, could trigger unpredictable immune reactions in patients. 2) ChR2 has a relatively high permeability to calcium, which might be toxic over the long term. 3) The ChR2 response is inherently weak at natural light intensities as each captured photon can only activate a single protein. 4) Although, melanopsin is able to amplify light-signals by gating the activities of high-throughput enzymatic reactions, these enzymatic partners are not sufficiently available in inner retinal neurons. Therefore, the expression of melanopsin in ganglion cells and ON-bipolar cells does not elicit an amplification of the light signal sufficient to restore functional vision at natural light intensities. 5) Also, the regulatory mechanisms that naturally control protein activity through changes in turnover and modulation are absent when expressing foreign proteins.
The object of the current invention is to provide a light-sensitive chimeric protein, which, when expressed in inner retinal neurons, overcomes these deficiencies. That is, it is an object of the invention to provide a superior light-sensitive protein for the improvement and restoration of vision, particularly in patients with retinal photoreceptor degeneration. This chimeric protein will improve or restore light-sensitivity to a higher extent compared to the light-sensitivity that is obtainable by proteins proposed in the state of the art. Further objects of the invention include the genetic information encoding the chimeric light-sensitive protein and methods of expressing this chimeric protein in living cells and organisms. Yet further objects of the invention include the expression of the genetic information encoding the chimeric light-sensitive protein in inner retinal cells in vivo for therapeutic treatment and biomedical products comprising the light-sensitive protein or genetic information encoding the chimeric protein.